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Ekaterina Papadopoulou and Rebecca Grumet

The Cucurbitaceae family is noted for a diversity of sex expression phenotypes. Typically, a phase of male flowers precedes either female or bisexual flower production. Sex determination of individual flowers is regulated by a combination of genetic, environmental, and hormonal factors. Ethylene, auxins, and gibberellins have all been shown to influence flower sex expression in cucurbits. Ethylene, which promotes femaleness, plays a predominant role. In this study, we tested whether brassinosteroids (BR), a more recently identified class of plant hormones, also influences cucurbit sex expression. Applied epi-brassinolide (epi-BL) caused a significant decrease in time of appearance of the first female flower on monoecious cucumber plants, and increased total female flowers on the main stem. Increasing concentrations had a stronger effect. Of the three species tested, cucumber, melon and zucchini, cucumber was the most responsive to BR. Application of epi-BL also caused an increase in ethylene production by cucumber and zucchini seedlings, suggesting that the BR effect may be mediated by ethylene. To investigate the possible relationship between BR and ethylene on sex expression, we identified the concentration of ethephon (5 ppm) that caused an increase in ethylene production comparable to that induced by 10 μm epi-BL (approximately two-fold). Treatment with 5 ppm ethephon was sufficient to increase femaleness of cucumber plants, but not zucchini plants, suggesting that the difference in response to epi-BL treatment may reflect differences in sensitivity to ethylene. Collectively, our results indicate that application of brassinosteroids to cucumber cause earlier and increased female flower production, and that the effects may be mediated, at least in part, by brassinosteroid-induced production of ethylene.

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Kaori Ando and Rebecca Grumet

Fruit rot induced by Phytophthora capsici Leonian is an increasingly serious disease affecting pickling cucumber (Cucumis sativus L.) production in many parts of the United States. The absence of genetically resistant cultivars and rapid development of fungicide resistance makes it imperative to develop integrated disease management strategies. Cucumber fruit which come in direct contact with the soil-borne pathogen are usually located under the canopy where moist and warm conditions favor disease development. We sought to examine whether variations in plant architecture traits that influence canopy structure or fruit contact with the soil could make conditions less favorable for disease development. As an extreme test for whether an altered canopy could facilitate P. capsici control, we tested the effect of increased row spacing and trellis culture on disease occurrence in the pickling cucumber `Vlaspik'. Temperature under the canopy was lowest in trellis plots, intermediate in increased spacing plots, and highest in control plots. Disease occurrence in the trellis plots was significantly lower than in other treatments, indicating that preventing fruit contact with the soil reduced disease occurrence. The effect of currently available variation in plant architecture was tested using nearly-isogenic genotypes varying for indeterminate (De), determinate (de), standard leaf (LL), and little leaf (ll) traits. Plants with standard architecture had higher peak mid-day temperatures under the canopy and greater levels of P. capsici infection; however, levels of disease occurrence were high for all genotypes. Screening a collection of ≈150 diverse cucumber accessions identified to serve as a representative sample of the germplasm, revealed variation for an array of architectural traits including main stem length, internode length, leaf length and width, and number of branches; values for `Vlaspik' were in the middle of the distribution. Plant architectures that may allow for more open canopies, including reduced branching habit and compact growth, were tested for disease incidence. One of the compact lines (PI 308916), which had a tendency to hold young fruit off the ground, exhibited lower disease occurrence. The reduced disease occurrence was not due to genetic resistance, suggesting that architecture which allows less contact of fruit with the soil could be useful for P. capsici control for pickling cucumber.

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Ekaterina Papadopoulou and Rebecca Grumet

The cucurbit family is noted for diversity in sex expression phenotypes.

Typically, a phase of male flowers precedes the appearance of female or hermaphrodite flowers. Sex determination of individual flowers is regulated by genetic, environmental, and hormonal factors. Ethylene, auxins, and gibberellins all influence flower sex, with ethylene, which promotes femaleness, playing a predominant role. In this study, we tested whether brassinosteroids, a more recently identified class of plant hormones, also influence cucurbit sex expression. Applied epi-brassinolide (epi-BL) caused a significant decrease in time of appearance of the first female flower on monoecious cucumber plants, and increased total female flowers on the main stem. Increasing concentrations had a stronger effect. Of the three species tested, cucumber, melon, and zucchini, cucumber was the most responsive. Application of epi-BL also caused an increase in ethylene production by cucumber and zucchini seedlings, suggesting that the BR effect may be mediated by ethylene. To investigate the possible relationship between BR and ethylene on sex expression, we identified the concentration of ethephon (5 ppm) that caused an increase in ethylene production comparable to that induced by 10 μm epi-BL (about two-fold). Treatment with 5 ppm ethephon was sufficient to increase femaleness of cucumber plants, but not zucchini plants, suggesting that the difference in response to epi-BL may reflect differences in sensitivity to ethylene. Collectively, our results indicate that application of brassinosteroids to cucumber cause earlier and increased female flower production, and that the effects may be mediated, at least in part, by brassinosteroid-induced increased production of ethylene.

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Guowei Fang and Rebecca Grumet

Zucchini yellow mosaic virus (ZYMV), a potyvirus, can cause major losses in cucurbit crops. With the goal of genetically engineering resistance to this disease we have engineered the ZYMV coat protein gene into a plant expression vector. The complete coat protein coding sequence, or the conserved core portion of the capsid gene, was attached to the 5' untranslated region of tobacco etch virus (TEV) in the pTL37 vector (Carrington et al., 1987, Nucl. Acid Res. 15:10066) The capsid constructs were successfully expressed by in vitro transcription and translation systems as verified by SDS-PAGE and ZYMV coat protein antibody. The constructs were then subcloned using polymerase chain reaction and attached to the CaMV 35 S transcriptional promoter on the CIBA-GEIGY pCIB710 plasmid. The constructs containing the CaMV 35S promoter, the 5' untranslated leader of TEV, and ZYMV coat protein sequences were then put between the Agrobacterium tumefaciens left and right borders in the pCIB10 vector and transferred to A. tumefaciens strain LBA4404 by triparental mating. These vectors are now being used to transform muskmelon and cucumber; resultant transgenic plants will be tested for ZYMV coat protein expression.

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Sue A. Hammar and Rebecca Grumet

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2 s -1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.

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Sue A. Hammar and Rebecca Grumet

We sought to develop efficient regeneratio nand transformation procedures for cucumber. Factors tested for regeneration included: hormone types and levels, genotype, explant source, and environmental conditions. Optimum regeneration was obtained using cotyledon pieces from 4 day old GY14A seedlings and culturing for 3 weeks under cool white lights (30-40 uE-2 s -1) on MS medium supplemented with 1.0 mg/l 2,4-D, 0.5 mg/l BA, 0.3 mg/l ABA, 30 g/l sucrose, 1 g/l MES, and 3.07 g/l Scott gelrite. Shoots developed via somatic embryogenesis ca. 2 wk after explants were transferred to MS supplemented with 20 g/l sucrose, 1 g/l MES, and 4.37 g/l gelrite. Ca. 80% of the explants produce shoots, 1/3-1/2 of which produce rooted plantlets; total time from explant to rooted plantlet is ca. 8 wks. Transformation experiments utilized Agrobacterium tumefaciens strains LBA4404 bearing the CIBA-GEIGY pCIB10 vector with a selectable marker gene for kanamycin resistance. Optimal conditions include 45 mg/l kan, 10 min inoculation and 3 day co-cultivation. Preliminary evidence suggests that tobacco nurse cultures increase transformation efficiency. Transgenic plants were confirmed by Southern or dot blot analysis.

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R.C. Yadav and Rebecca Grumet

Tendrils were used as an alternate tissue source to determine the ploidy level of transgenic melon (Cucumis melo L.) plants. Young tendril tips were prepared for chromosome visualization using standard root tip preparation techniques. Well-spread chromosome preparations were obtained. Tendrils were found to be as amenable to chromosome visualization as root tips. The transformed melon plants, representing three transgenic melon lines, were tetraploid. The tendril technique also was applied successfully to several other tendril-producing species: cucumber (C. sativus L.), pea (Pisum sativum L.), and grape (Vitis vinifera L.). This technique can be very useful for chromosome visualization in tendril-producing species, and can be especially valuable when root tips or anthers cannot be obtained easily.

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John Everard, Rebecca Grumet and Wayne Loescher

In celery, photosynthetic carbon partitioning between mannitol and sucrose is highly dependent on developmental (leaf age) and environmental (salt stress) factors. Mannose 6-phosphate reductase (M6PR) mediates a key step in mannitol biosynthesis and may regulate partitioning between sucrose and mannitol. We have constructed a cDNA library and have isolated M6PR-specific clones. Before library construction, poly(A)+ RNA, extracted from newly fully expanded leaves, was translated in vitro. A single polypeptide (35.1 kD), immunoprecipitated with M6PR-specific antisera, accounted for ≈5% of the total 35S incorporated into TCA-precipitated products. Parity between the molecular masses of the immunoprecipitated product and authentic M6PR indicated minimal posttranslational modification. The unidirectional primary library, constructed in UniZap XR vector (Stratagene), consisted of 1.53 million plaque forming-units (pfus) of which <0.4% were nonrecombinant, as estimated by “blue/white”' screening. After a single amplification, ≈0.14% of the 200,000 pfus screened with M6PR-specific antisera were identified as putative M6PR clones. Following two further rounds of screening and in vivo excision of the pBluescript phagemids their identity as full length M6PR clones was confirmed as follows: 1) IPTG-induced expression of M6PR activity in crude extracts; 2) IPTG-induced expression of a polypeptide that specifically interacted with M6PR antisera and with identical mobility (on SDS gels) to authentic M6PR; 3) 100% sequence homology to an internal peptide from a tryptic digest of purified M6PR. Based on these criteria, we conclude that we successfully cloned M6PR. The sequence is similar to several reductases from both plants and animals including an aldose 6-phosphate reductase from apple. Supported by USDA-NRI grant 940-1439.

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Thanda Wai, Jack Staub and Rebecca Grumet

TMG-1 is resistant to ZYMV and WMV-2, two closely related potyviruses. Resistance to ZYMV is due to a single recessive gene (Provvidenti, 1987); however, two recessive genes appear to confer resistance to WMV-2. We sought to further characterize the resistances by studying possible linkage relationships with physiological, morphological, electromorphic, and phytopathological markers. TMG-1, WI-2757 (an inbred line susceptible to both viruses), and their F2 progeny were screened for various single gene characters that differ between the two parents. Linkages reported in the literature were also observed in this study: (1) between bitterfree (bi) and female (F), and (2) between numerous spine (ns), small spine (ss), and tuberculate (Tu). New linkages detected were between: (1) resistance to WMV-2 and F, (2) resistance to WMV-2 and ZYMV, and (3) possibly resistance to ZYMV with fusarium and ns.